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Structure and functional characterization of the periplasmic N‐terminal polypeptide domain of the sugar‐specific ion channel protein (ScrY porin)
Author(s) -
Michels Jenny,
Geyer Armin,
Mocanu Viorel,
Welte Wolfram,
Burlingame Alma L.,
Przybylski Michael
Publication year - 2002
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.2760102
Subject(s) - chemistry , porin , trimer , circular dichroism , protein structure , coiled coil , electrospray ionization , protein secondary structure , crystallography , antiparallel (mathematics) , periplasmic space , dimer , biochemistry , stereochemistry , bacterial outer membrane , mass spectrometry , chromatography , physics , organic chemistry , escherichia coli , quantum mechanics , magnetic field , gene
The structure of the sucrose‐specific porin (ScrY) from Salmonella typhimurium has been elucidated by X‐ray crystallography to consist of 18 antiparallel β‐strands, associated as a trimer complex similar to ion‐transport channels. However, the 71‐amino‐acid‐residue N‐terminal periplasmic domain was not determined from the crystal structure due to the absence of sufficient electron density. The N‐terminal polypeptide contains a coiled‐coil structural motif and has been assumed to play a role in the sugar binding of ScrY porin. In this study the proteolytic stability and a specific proteolytic truncation site at the N‐terminal domain were identified by the complete primary structure characterization of ScrY porin, using MALDI mass spectrometry and post‐source‐decay fragmentation. The secondary structure and supramolecular association of the coiled‐coil N‐terminal domain were determined by chemical synthesis of the complete N‐terminal polypeptide and several partial sequences and their spectroscopic, biophysical, and mass spectrometric characterization. Circular dichroism spectra revealed predominant α‐helical conformation for the putative coiled‐coil domain comprising residues 4–46. Specific association to both dimer and trimer complexes was identified by electrospray ionization mass spectra and was ascertained by dynamic light scattering and electrophoresis data. The role of the N‐terminal domain in sugar binding was examined by comparative TR‐NOE‐NMR spectroscopy of the complete ScrY porin and a recombinant mutant, ScrY(Δ1–62), lacking the N‐terminal polypeptide. The TR‐NOE‐NMR data showed a strong influence of ScrY porin on the sugar‐binding affinity and suggested a possible function of the periplasmic N terminus for supramolecular stabilization and low‐affinity sugar binding.